The overall goals of this proposal are to determine the extent to which human embryonic stem cell (hESC)- or induced pluripotent stem cell (iPSC)-derived neural progenitor cell (NPC) grafts improve recovery after experimental stroke, and to begin addressing the critical safety and efficacy questions necessary for eventual human stroke therapy. hESCs offer many advantages as a source of NPCs for regenerative therapy, such as a readily available supply, vast differentiation potential and ease of genetic manipulation. iPSCs offer the added advantage of autologous grafting that obviates the need for immunosuppression. For either source, however, the ideal donor cell types and developmental states required to achieve CNS regeneration are poorly understood. Whether NPC grafting restores function after stroke and the mechanisms by which it might do so also are unknown. We have developed methods to enrich for specific NPC populations derived from hESCs, and have generated NPCs and multiple neuronal subtypes using iPSCs derived from human fibroblasts. Using these techniques, we propose to test the following hypotheses: 1) purified populations of multipotent NPCs (mpNPCs) and neuronal restricted precursors (NRPs) can be derived from hESCs or from human somatic cells via iPSCs. These populations will differ in their migration, differentiation and integration after transplantation into the intact or injured adult rat brain; and 2) grafting of mpNPCs or NRPs after experimental stroke will enhance functional recovery directly by neuronal replacement or by stimulating repair via endogenous NPCs. Specific Aims 1 and 2 are to purify and characterize specific NPC populations using promoter-based reporter or cell surface antigen-based selection. Aim 3 is to examine the behavior of these NPC populations after grafting into neurogenic and non-neurogenic regions of the adult rat brain, and Aim 4 is to examine the influence of hESC- and iPSC (human and rat)-derived NPC grafts on recovery after experimental stroke. Progress in these aims will advance our knowledge of how graft factors influence the capacity of hESC- or iPSC-derived NPCs to repair the injured brain and promote recovery after experimental stroke, and will provide insight into the untapped reparative potential and possible risks of these therapies. PUBLIC HEALTH RELEVANCE: Stroke is a common and potentially devastating neurological disorder with no proven regenerative therapies. This proposal aims to derive neural stem cells (NSCs) from human embryonic stem cells or reprogramming of human adult skin cells, and to use a stroke model to identify the optimal NSC grafts for brain reparative stroke therapy. Progress in this area offers advances toward novel cell-based restorative therapies for stroke and other brain insults.